Biology subjects

Coll, R. C.

Publications and source records attributed to Coll, R. C..

7 recordsLinked to original sources

bioRxiv2025

NLRP3 is a thermosensor that is negatively regulated by high temperature

Inflammation is an essential response to infection and injury, but unregulated inflammation is damaging and must be limited by negative feedback signalling. Inflammasome signalling drives local inflammation and systemic responses like fever. However, our understanding of how inflammasome signalling is negatively regulated is limited. NLRP3 is activated by a vast number of stimuli and senses perturbations of cytoplasmic homeostasis. As temperature is a fundamental environmental stressor, we hypothesised that NLRP3 inflammasome signalling would be sensitive to increased temperatures and so we investigated the effects of high temperatures on NLRP3 in macrophages. Short-term incubation at high fever range temperatures significantly inhibits NLRP3 activation, while secretion of the inflammasome-independent cytokines TNF and IL-6 are much less affected. High temperature blocks NLRP3 inflammasome formation in a transcription-independent manner, and NLRP3 is highly sensitive to temperature-mediated inhibition relative to the NLRC4, AIM2, and NLRP1 inflammasomes. Using cellular assays and molecular simulations we show that the effect of high temperature on NLRP3 is protein intrinsic. NLRP3 activation is associated with a decrease in the thermal stability of the protein and multiscale molecular dynamics simulations identified a peptide in the C-terminal of the FISNA domain (COFI) that is highly flexible and undergoes a significant conformational shift at high temperature. Cellular assays demonstrate that the COFI regulates NLRP3 stability and is required for activation. Furthermore, mice exposed to high temperature display attenuated inflammatory cytokine production upon in vivo LPS challenge. Our studies thus reveal that high temperatures associated with fever limit NLRP3 activity and identify a novel role for NLRP3 as a protein thermosensor.

immunology↗

bioRxiv2025

Induced pluripotent stem cell-derived macrophages as a model for human inflammasome signaling

Macrophage models are a mainstay of inflammasome research, however current human in vitro macrophage models have significant limitations. Here we generate induced pluripotent stem cell (iPSC)-derived macrophages (iMacs) to study inflammasome signaling and benchmark them with human monocyte-derived macrophages (HMDMs). We confirm that iMacs express high levels of macrophage markers and are highly phagocytic. Whole cell proteomics analysis shows that iMacs express many inflammasome sensors and related proteins, and in functional assays iMacs respond to multiple inflammasome stimuli. The NLRP3 inflammasome is strongly activated in iMacs and we find that nigericin alone activates NLRP3. The non-canonical inflammasome does not require a priming step in iMacs as caspase-4 is constitutively expressed. High levels of NAIP/NLRC4 inflammasome activation are also observed in response to needle toxin. Finally, unlike HMDMs, iMacs activate NLRP1. Therefore, we demonstrate that iMacs are a physiologically relevant and highly tractable model to study human inflammasome signaling and regulation. MotivationiPSC-derived macrophages (iMacs) are functionally, transcriptionally, and phenotypically similar to primary human macrophages. iMacs therefore offer new opportunities to study inflammasome activity in a human macrophage model, but to date they have not been widely used. In this study, we describe a protocol to differentiate and characterize iMacs. We then describe how to activate a range of different inflammasomes within these cells and assess the inflammasome response by measuring pyroptosis, cytokine release, ASC speck formation, and processing of inflammasome-related proteins. We also benchmark iMac responses with the current gold standard primary human monocyte derived macrophage model.

immunology↗

bioRxiv2025

Inhibition of NLRP3 by a CNS-penetrating indazole scaffold

Low-grade inflammation is a hallmark of ageing and a key cause of age-related impairments and diseases1. The NOD-like receptor NLRP3 senses a variety of danger signals and environmental insults, resulting in pro-inflammatory response, inflammasome formation and pyroptosis2,3. Its aberrant activation has been linked to many acute and chronic diseases ranging from atherosclerosis to Alzheimers disease and cancer, making NLRP3 an attractive therapeutic target4,5. Here we report the discovery, characterization, and structure of an indazole-based NLRP3 antagonist, BAL-1516, which potently inhibits inflammasome formation in monocytes and microglia. The cryo-electron microscopy structure of BAL-1516 bound to NLRP3 reveals a previously undescribed compound binding site at a surface groove of the nucleotide-binding domain with contacts to the FISNA and WHD subdomains. The characteristic feature of BAL compound binding is the formation of three hydrogen bonds to the peripheral {beta}-strand of the triple-ATPase; two from the indazoles nitrogen atoms and a third from the compounds linker region. Additional phenyl and thiazole moieties render the compound hydrophobic, allowing excellent blood-brain barrier penetration. The compound binding site is highly specific for NOD-like receptors, and the optimized compound BAL-1516 is able to directly bind mouse NLRP3 despite two conservative residue changes in the binding interface. The BAL compounds represent a first-in-class family of NLRP3 inhibitors, providing a broad design space, including covalent and degradative properties, for the development of NLRP3-directed therapeutics. The innate immune system contains cytosolic proteins that sense cellular stress caused by bacterial, viral and fungal infections or sterile inflammation, to control cellular integrity2. NLRP3 is a well-studied member of the nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) that is involved in the activation of the inflammasome, a multiprotein complex that mediates inflammation6. Upon detection of stress or pathogen-associated signals, NLRP3 triggers the activation of caspase-1, which leads to the production of pro-inflammatory cytokines such as IL-1{beta} and IL-18, driving inflammatory responses and ultimately pyroptotic cell death. In the context of neuroinflammation, NLRP3 plays a significant role in the pathogenesis of various neurodegenerative diseases, including Alzheimers disease, Parkinsons disease, and multiple sclerosis7. Research into targeting NLRP3 signalling with CNS-penetrating molecules holds potential for developing therapeutic strategies to alleviate neuroinflammatory conditions and to slow the progression of neurodegenerative diseases.

immunology↗

bioRxiv2025

Plasma membrane rather than endosomal Gq signaling drives transcriptional activity by the viral chemokine receptor US28 in glioblastoma

US28 is a human cytomegalovirus-encoded chemokine receptor homologue that has high agonist-independent activity, internalizes constitutively, and plays an oncomodulatory role in glioblastoma. As G protein signaling was originally believed to strictly occur at the plasma membrane, it has been assumed that US28s constitutive Gq/11 signaling is mediated by a minor population at the plasma membrane. However, accumulating evidence shows that some GPCRs activate G proteins from intracellular organelles, such as endosomes. Importantly, endosomal rather than plasma membrane G protein signaling has been associated with transcriptional activity. Here, we demonstrate that the endosomal US28 population robustly activates Gq/11, and thus, provides the major contribution of Gq/11 signaling. Surprisingly, US28 signaling at the plasma membrane rather than from endosomes primarily drives upregulation of gene expression involved in cell proliferation and inflammatory responses that are associated with glioblastoma and cancer. Our findings highlight the crucial role of receptor signaling location in cellular responses.

cell biology↗

bioRxiv2024

Discovery of a Potent and Selective Inhibitor of Human NLRP3 with a Novel Binding Modality and Mechanism of Action

The NLRP3 inflammasome is an intracellular protein complex that causes inflammation via the release of IL-1{beta} and pyroptosis. NLRP3 activation is associated with many age-related inflammatory diseases, and NLRP3 inhibition is a promising therapeutic strategy. We previously performed a DNA encoded library screen to identify novel NLRP3 binding molecules. Herein we describe the characterization of BAL-0028 as a potent and specific inhibitor of NLRP3 signaling. Notably, BAL-0028 is a poor inhibitor of mouse NLRP3 but inhibits human and primate NLRP3 with nanomolar potency. Using cellular and biochemical analyses we demonstrate that BAL-0028 binds to the NLRP3 NACHT domain at a site that is distinct from the MCC950 binding pocket. Using humanized NLRP3 mice we show that a derivative of BAL-0028 inhibits NLRP3 activation in vivo in a peritonitis model. Finally, we demonstrate that BAL-0028 inhibits select hyperactive NLRP3 mutations associated with autoinflammatory diseases more potently than does MCC950. BAL-0028 thus represents a new modality for NLRP3 inhibition in inflammatory diseases. SUMMARYNLRP3 is a target for anti-inflammatory therapies and can be inhibited by the tool compound MCC950. We describe the characterization of a new small molecule inhibitor of NLRP3 BAL-0028 that has a distinct mechanism of action and binding site.

immunology↗

bioRxiv2024

Butyrate and propionate are microbial danger signals that activate the NLRP3-inflammasome in human macrophages in the presence of TLR stimulation

Short chain fatty acids (SCFAs) are immunomodulatory compounds produced by the microbiome through fermentation of dietary fibre. Although they are generally considered beneficial for gut health, patients suffering from inflammatory bowel disease (IBD) have shown poor tolerance to fibre-rich diets, suggesting that SCFAs may have contrary effects under inflammatory conditions. To investigate this, we examined the effect of SCFAs on human macrophages in the presence of toll-like receptor agonists. In contrast to their anti-inflammatory effects under steady state conditions, we observed that the SCFAs butyrate and propionate triggered the activation of the NLRP3 inflammasome when added in conjunction with TLR agonists. Mechanistically, butyrate and propionate activated NLRP3 by inhibiting HDACs 1-3 and 10, leading to an uneven distribution of histone hyperacetylation that resulted in alterations in the transcriptome. Specifically, there was a lack of hyperacetylation at the loci of the CFLAR and IL10 genes, two important inhibitors of NLRP3 inflammasome activation. The concurrent loss of transcription and protein expression of cFLIP and IL-10 enabled caspase-8-dependent NLRP3-inflammasome activation. SCFA-driven NLRP3 activation did not require potassium efflux and did not result in cell death but rather triggered hyperactivation and IL-1{beta} release. Our findings demonstrate that butyrate and propionate are bacterially-derived, viability-dependent danger signals (vita-PAMPs) that regulate NLRP3 inflammasome activation through epigenetic modulation of the inflammatory response. SummaryUnder inflammatory conditions, SCFAs are bacterially-derived, viability-dependent danger signals that, through HDAC inhibition and epigenetic modification, prevent expression of the anti-cell death gene cFLIP to trigger activation of the NLRP3 inflammasome.

immunology↗

bioRxiv2023

The Achromobacter Type 3 secretion system drives pyroptosis and immunopathology via independent activation of NLRC4 and NLRP3 inflammasomes

Achromobacter species are newly recognized opportunistic, pro-inflammatory Gram-negative pathogens in immunocompromised individuals, but how they interact with the innate immune system to drive inflammation is poorly understood. We created sctV (Type 3 Secretion System baseplate) mutants in three Achromobacter clinical isolates from two species and showed that all three required the T3SS to induce cell death in human macrophages. Mutating other critical T3SS components also abolished cell death, which was restored by genetic complementation. Cell death of Achromobacter-infected macrophages was contact-dependent, enhanced by bacterial internalisation, and caused by inflammasome-dependent pyroptosis (typified by Gasdermin-D cleavage and IL-1{beta} secretion). Macrophages deficient in the inflammasome sensors NLRC4 or NLRP3 underwent pyroptosis upon bacterial internalization but those deficient in both NLRC4 and NLRP3 did not, suggesting either sensor can mediate pyroptosis induction in a T3SS-dependent manner. Detailed analysis of the intracellular trafficking of one isolate indicated that the intracellular bacteria reside in an acidic LAMP-1/dextran-positive membrane compartment. Using an intranasal mouse infection model, we observed that Achromobacter damages lung structure and causes severe illness, contingent on a functional T3SS. Together, we demonstrate that Achromobacter species can survive phagocytosis by macrophages and promote macrophage cell death and inflammation by redundant mechanisms of pyroptosis induction.

microbiology↗